Abnormal neuronal development can lead to a wide array of psychiatric disorders. Mutations disrupting protein coding genes have been found to cause some of these disorders but a large number of them still remain unsolved. A variety of molecular and clinical data suggests that mutations in gene regulatory sequences could be a major contributor to these disorders. However, only a few causal regulatory mutations have been found to date. This is primarily because functional regulatory elements are difficult to identify, particularly in mixed cell populations such as the developing brain. In addition, these elements are difficult to functionally characterize in a high-throughput manner in these cell types. To address these challenges, we propose to use novel single-cell genomic technologies along with massively parallel reporter assays (MPRAs) in human primary cells and organoids to characterize thousands of brain development associated genes, regulatory elements and pathways. First, using single cell RNA-seq (scRNA-seq) and ATAC-seq (sci-ATAC-seq) across multiple cortical areas and subcortical regions of developing human brain at three development stages, we will generate a comprehensive map of genes, regulatory elements and networks involved in human brain development (Aim 1). Next, we will use similar techniques (scRNA-seq and sci-ATAC-seq) on human cerebral organoid cultures derived from induced pluripotent stem cells (iPSCs). We will compare regulatory programs in organoid cells to cells present during normal human brain development. To assess the contribution of key transcription factors involved in psychiatric disorders to gene regulatory pathways in the developing brain, we will use genome editing on the same genetic background to create heterozygous loss-of-function mutations in key transcription factors involved in psychiatric disorders and assess their effects on gene expression (scRNA-seq) and gene regulation (sci- ATAC-seq) (Aim 2). Finally, we will functionally characterize over 37,500 candidate enhancers and nucleotide variants within them using a lentiviral-based MPRA (lentiMPRA) in disease-relevant cell types purified from human primary cells and organoids. Several of these sequences will also be assayed in organoids lacking key transcription factors deleted in Aim 2 to test the importance of these genes to regulatory activity and to identify interactions with regulatory variants (Aim 3). Data from all aims will be used to build predictive models of gene expression and enhancer activity as a function of regulatory sequences, which will be used to design lentiMPRA libraries and iteratively improve models using results from initial libraries. Combined our project will use cutting- edge techniques such as scRNA-seq, sci-ATAC-seq and MPRA coupled with advanced computational analyses to significantly increase the number of functionally characterized human brain developmental regulatory elements and how their activity changes in the presence of disease associated mutations to shed light on the genetic basis for psychiatric disorders.